Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses

Summary

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-γ (IFN-γ) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD₅₀ dose of the pathogen.

Abstract

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-γ by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen^1-3. In addition, IFN-γ production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection⁴. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-γ production have an impact on IFN-γ responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-γ production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-γ responses to L. monocytogenes and survival of mice from this infection.

Introduction

IFN-γ is a cytokine that is crucial for mediating immunity against intracellular pathogens and for controlling tumor growth⁵. The importance of this cytokine in bacterial resistance is evident in the observation that humans with mutations in the IFN-γ signaling pathway are highly susceptible to infection with mycobacteria and salmonellae⁶. Similarly, mice deficient in either IFN-γ or the IFN-γ receptor exhibit defects in resistance to mycobacteria^7-9 and other intracellular pathogens including L. monocytogenes^10,11, Leishmania major¹², Salmonella typhimurium¹³

Protocol

Safety Statement

This protocol describes infection of mice with live L. monocytogenes. The pathogen is handled safely under BSL2 conditions by trained personnel who are not immunocompromised. Immunocompromised people include pregnant women, the elderly and individuals who are HIV-infected or have chronic conditions that require treatment with immunosuppressive therapy. Personnel should don a protective lab coat or gown, gloves, mask, and eye protection while handling infected samples. The work described herein was performed under BSL2 conditions under a certificate (#32876) that was issued by the University Health Network (UHN) Biosa....

Results

Figure 3 presents some typical flow cytometry staining of IFN-γ in splenic NK and NKT cells at 24 hr post-infection with 10⁵ CFU of the pathogen. This figure also illustrates the gating strategy for the staining panel described in Table 2. Figure 4 shows some representative data that were obtained in one experiment where male mice were treated with the PPARα antagonist IS001 or vehicle control, infected with 10^5<.......

Discussion

Here we describe a protocol of how to carry out a basic experimental infection with the EGD strain of L. monocytogenes²⁵ in male or female C57BL/6J mice. This protocol was set up for the purpose of studying the effect of a novel small molecule IS001 on IFN-γ production by innate and adaptive lymphocytes in vivo³¹. By monitoring bacterial clearance and survival post-infection, insights were gained into how these changes in IFN-γ impacted the host's ability to control t.......

Materials

Name	Company	Catalog Number	Comments
Brain Heart Infusion Broth, Modified	BD	299070	any brand should be appropriate
Agar	BD	214010	any brand should be appropriate
Triton X-100	Sigma-Aldrich	X100	any brand should be appropriate
1xPBS	Sigma	D8537	any brand should be appropriate
TissueLyser II	Qiagen	85300	any brand should be appropriate
Ammonium Chloride (NH3Cl)			any brand should be appropriate
KHCO3			any brand should be appropriate
Na2EDTA			any brand should be appropriate
RPMI 1640	Gibco	22400089	any brand should be appropriate
Fetal Bovine Serum	Gibco	12483	Before use, heat-inactivate at 56 °C for 30 min
L-glutamine	Gibco	25030	any brand should be appropriate
Non-essential amino acids	Gibco	11140	any brand should be appropriate
Penicillin/Streptomycin	Gibco	15140	any brand should be appropriate
GolgiStop Protein Transport Inhibitor (containing Monensin)	BD	554724	Use 4 μl in 6 ml cell culture
16% Paraformaledehye	Electron Microscopy Sciences	15710	Dilute to 4% PFA in ddH20 or 1xPBS
10 x Perm/Wash buffer	BD	554723	Dilute 10x in ddH20
Fc block, Anti-Mouse CD16/CD32 Purified	eBioscience	14-0161	Dilute 1:50
Fixable Viability Dye eFluor 506	eBioscience	65-0866	Dilute 1:1000 (we have also used viability dyes from Molecular Probes)
anti-Mouse CD4-PE-Cy5 (GK1.5)	eBioscience	15-0041	Manufacturer recommends a certain test size; however this should be titrated before use.
anti-Mouse CD8-FITC (53-6.7)	eBioscience	11-0081	Manufacturer recommends a certain test size; however this should be titrated before use.
PBS57/mCD1d tetramer-APC	NIH Tetramer Core Facility	N/A	Obtained as a gift from the facility
anti-Mouse TCRβ-PE-Cy7 (H56-597)	eBioscience	25-5961	Manufacturer recommends a certain test size; however this should be titrated before use.
anti-Mouse NKp46-APC-eFluor780 (29A1.4)	eBioscience	47-3351	Manufacturer recommends a certain test size; however this should be titrated before use.
anti-Mouse CD45 PE-Cyanine7 (30-F11)	eBioscience	25-0451	Manufacturer recommends a certain test size; however this should be titrated before use.
anti-Mouse IFN gamma-PE (XMG1.2)	eBioscience	12-7311	Manufacturer recommends a certain test size; however this should be titrated before use.
OneComp eBeads	eBioscience	01-1111	Manufacturer recommends a certain test size; however this should be titrated before use.
Mouse IFN gamma ELISA kit	eBioscience	88-7314	Used for measuring the interferon gamma in the culture supernatant
50 mL vented tubes for culture			Used for culturing the bacteria, any brand should be appropriate
1.5 ml microcentrifuge tubes			any brand should be appropriate
bacterial petri dishes			any brand should be appropriate
2 ml cyrovials			any brand should be appropriate
UV spectrometer			any brand should be appropriate
safety engineered needles			any brand should be appropriate
C57BL6/J	Jackson laboratories	Stock#000664	Order for arrival at 7 wks
Bleach			For decontamination
70% Ethanol			For decontamination
Glass beads			any brand should be appropriate
Centrifuge			rotor, buckets, bucket covers.
Microcentrifuge			any brand should be appropriate
Sterile Glycerol			any brand should be appropriate
Pipette Tips			any brand should be appropriate
Pipette			any brand should be appropriate
Surgical instruments			any brand should be appropriate
70 micron strainers			any brand should be appropriate
3 ml syringe			any brand should be appropriate
Pipette gun			any brand should be appropriate
Filtration Units			any brand should be appropriate
Trypan Blue			Dilute 1 to 9 in ddH20, any brand should be appropriate
Hemocytometer			any brand should be appropriate
Round bottomed plates			any brand should be appropriate
FACs tubes	BD
BD LSR II	BD		Any flow cytometer could be used for acquisition that has an appropriate laser configuration and filter set to discriminate the fluorochormes
Flowjo software	Treestar		Used for data analysis. Other types of data analysis software will also be appropriate
Multichannel pipettor (0-300 µl)	Eppendorf		Used for washing cells and adding antibodies during flow cytometry staining
Acetic Acid			Used for washing glass beads, any brand should be appropriate
Microbank Bacterial Preservation System	Pro-lab Diagnositics		Used as an alternative to glycerol stocks for long-term storage of bacteria